The goal of this project is to define the molecular mechanisms involved in the replication of enveloped RNA viruses and in particular, to understand the factors which influence the regulation and expression of viral genetic information. Studies are being carried out with the murine leukemia virus system. Current interest is focused on the process of reverse transcription in an effort to correlate genetic structure with enzymatic function. The genetic defect in a nonconditional pol mutant has now been identified. This mutant produces a truncated reverse transcriptase with reduced levels of enzymatic activity. Using recombinant DNA technology, a molecular clone of the entire viral genome was obtained and the mutation was localized to a 400 base pair region near the middle of the pol gene. Sequence analysis of this region has demonstrated that the mutant genome contains a one-base insertion which brings three TGA codons into phase and results in premature termination of translation at a position consistent with the observed size of the mutant enzyme. Correlation of the precise location of the mutation with the known mutant phenotype has led to a map for the genetic organization of the MuLV pol gene and prediction of a virus-encoded protease at the 5'end of pol. In addition, the active sites for polymerase and RNase H can now be localized to the N-terminal domain of reverse transcriptase. Studies on "enzyme pausing" in endogenous MuLV reverse transcription have continued. Computer analysis of intermediate bands indicates that the location of pause sites correlates with the presence of a set of C-rich consensus sequences clustered within predicted multibranch loop structures which can be formed by the viral RNA template. Attempts to express the MuLV pol gene in E. coli bacteria are also in progress.